The human DNA tumor virus adenovirus encodes two transforming genes, E1A and E1B, which cooperate to transform primary rodent cells. The E1A gene products efficiently stimulate cell proliferation but fail to transform cells due to the induction of programmed cell death (apoptosis). Induction of apoptosis by E1A is mediated by the product of the p53 tumor suppressor gene indicating that p53 can function as a tumor suppressor by initiating cell death. Expression of the E1B gene or the human bcl-2 proto-oncogene, blocks E1A-induced p53-dependent apoptosis to produce transformation. Thus, deregulation of cell growth control must be coupled to suppression of an intrinsic cell suicide response for the efficient transformation of primary cells. The E1B gene encodes two unique l9kDa and 55kDa proteins, both of which block apoptosis by interfering with the function of p53. The E1B 55K protein binds to and inactivates p53 directly, whereas the.mechanism utilized by the E1B l9K protein to inhibit p53-mediated apoptosis is not yet known. Overexpression of the human Bcl-2 protein will similarly block p53-dependent apoptosis and will substitute for the E1B l9K protein in all functional assays. It is highly probable that the E1B l9K protein represents the viral equivalent of Bcl- 2. I intend to determine how the E1B l9K and Bcl-2 proteins act at the biochemical level to block p53-dependent apoptosis. An essential aspect of this work is to identify the cellular proteins with which the E1B l9K and Bcl-2 proteins interact. We have evidence that the E1B l9K protein does not bind p53 directly, indicating that the l9K protein is an indirect modifier of p53 function. First, the cellular proteins which interact with the E1B l9K protein will be identified. Second, I will determine how the interaction prevents the apoptotic activity of p53. The two-hybrid system in yeast will be utilized to identify cellular proteins which interact with the E1B l9K protein. This work will complement the ongoing NIH funded project to identify l9K binding proteins by strictly biochemical means. The goal of these aims is to develop a complete mechanism by which the cell controls the activity of p53. It is becoming increasingly apparent the regulation of death may be as important as regulation of proliferation, as an intracellular defense against viral infection and cancer. Determining how apoptosis is induced by p53 and how the transforming proteins of DNA tumor viruses intervene and subvert this process is of fundamental importance to understanding the cause and prevention of cancer.